SUMMARY Cellular behavior is modulated by environmental signals including nutrients, osmotic stress, hormones and neurotransmitters. These signals can vary considerably in dose, duration, and directionality. This proposal seeks to (i) quantitatively measure the yeast pheromone response pathway, both in time and space, (ii) devise computational models that describe the observed behaviors, and (iii) test the validity of each model through further experimentation. The over-arching hypothesis is that mathematical techniques developed for studying dynamical systems can explain how pathway components interpret and translate spatial cues outside the cell to evoke appropriate responses inside the cell. The focus will be on proteins that modulate the time-dependent behaviors of the pheromone pathway in yeast, and in particular how these modulators contribute to proper signal transduction. Models will be tested experimentally at the molecular and cellular level, and include the use of an innovative gradient flow chamber. There are three specific aims, focused on three different proteins acting at three distinct steps in the pathway. All three proteins are required for gradient-sensing activity. Aim 1 will investigate time-dependent regulation by the scaffold protein Ste5. This aim will test the hypothesis that Ste5 modulates signaling by imposing a delay between each of two phosphorylation events needed for full activation of a MAP kinase Fus3. Aim 2 will investigate time- and space-dependent regulation by the RGS protein Sst2. This aim will test the hypothesis that Sst2 functions as a scaffold protein that coordinates G protein activation (by the receptor) and inactivation (of the G protein), and thereby orients the cell towards a gradient stimulus. Aim 3 will investigate time- and space-dependent regulation by the pheromone protease Bar1. This aim will test the hypothesis that Bar1 remodels the gradient, and thereby coordinates the behavior of two cells responding to the same stimulus. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Proper cell function requires the ability to detect and respond appropriately to hormones, neurotransmitters and drugs. The cellular machinery responsible for signal transmission is conserved from humans to yeast. This project uses multi-disciplinary approaches, including biological experiments, microfabricated growth chambers and computer simulations, to establish how the direction and strength of a stimulus are interpreted by the cell. The broader objective is to understand the role of spatial and temporal information in cell signaling, and eventually predict what drug treatments will be most effective in combating human diseases.